Polyampholytes for delivering polyions to a cell

ABSTRACT

An polyampholyte is utilized in a condensed polynucleotide complex for purposes of nucleic acid delivery to a cell. The complex can be formed with an appropriate amount of positive and/or negative charge such that the resulting complex can be delivered to the extravascular space and may be further delivered to a cell.

[0001] This Application is a Continuation-In-Part of Ser. No. 09/000,692filed on Dec. 30, 1997 and related to provisional application No.60/174,132 filed on Dec. 31, 1999.

FIELD OF THE INVENTION

[0002] The invention relates to compounds and methods for use inbiologic systems. More particularly, polyions are utilized for modifyingthe charge (“recharging”) of particles, such as molecules, polymers,nucleic acids and genes for delivery to cells.

BACKGROUND

[0003] Polymers are used for drug delivery for a variety of therapeuticpurposes. Polymers have also been used in research for delivery ofnucleic acids (polynucleotides and oligonucleotides) to cells, theprocess is one step in reaching a goal of providing therapeutc processes(gene therapy). One of the several methods of nucleic acid delivery tothe cells is the use of DNA-polyion complexes. It has been shown thatcationic proteins like histones and protamines or synthetic polymerslike polylysine, polyarginine, polyornithine, DEAE dextran, polybrene,and polyethylenimine may be effective intracellular delivery agentswhile small polycations like spermine are ineffective.

[0004] In terms of intravenous injection, DNA must cross the endothelialbarrier and reach the parenchymal cells of interest The largestendothelia fenestrae (holes in the endothelial barrier) occur in theliver and have an average diameter from 75-150 nm. The trans-epithelialpores in other organs are much smaller, for example, muscle endotheliumcan be described as a structure which has a large number of small poreswith a radius of 4 nm, and a very low number of large pores with aradius of 20-30 nm. The size of the DNA complexes is also important forthe cellular uptake process. After binding to the target cells theDNA-polycation complex should be taken up by endocytosis.

[0005] Applicants have provided a process for delivering a compoundacross the endothelial barrier to the extravascular space and then to acell.

Summary

[0006] Described in a preferred embodiment is a process for enhancingdelivery of a polyion to a cell, comprising the formation of a complexof polyampholyte and polyion Then, delivering the complex into a cell.

[0007] In another preferred embodiment, we describe a process forextravasation of a complex. The process comprises the formation of acomplex of polyampholyte and polyion. Then, inserting the complex into avessel and delivering the complex to an extravascular space.

[0008] Reference is now made in detail to the preferred embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 DNA interactions with pC-pA block polyampholyte: binding(pA low charge density) and replacement (pA high charge density).

[0010]FIG. 2 Isolation of lPEI-pGlu polyampholyte using Example 1reaction mixture.

[0011]FIG. 3 Transfection of HUH7 cells using lPEI-pGlu polyampholyteusing Example 1 reaction mixture.

[0012]FIG. 4 Transfection of HUH7 cells using lPEI-pMAA polyampholyteusing Example 1 reaction mixture.

[0013]FIG. 5 Sepharose 4B-CL chromatography of rhodamine-labeled pGluand lPEI-pGlu polyampholyte.

[0014]FIG. 6 Luciferase expression in HUH7 cells in vitro in 100% bovineserum aided by covalent lPEI-pGlu polyampholyte as compared to lPEI-pGlunon-covalent mixture and lPEI alone.

DETAILED DESCRIPTION

[0015] Polyampholytes are copolyelectrolytes containing both polycationsand polyanions in the same polymer. In aqueous solutions polyampholytesare known to precipitate near the isoelectric point and formmicelle-like structures (globules) at the excess of either charge. Suchglobules maintain tendency to bind other charged macromolecules andparticles (see R R Netz, J F Joanny, Macromolecules, 31, 5123-5141(1998)).

[0016] In provisional application Ser. No. 60/093,153 we described genetransfer activity which can be substantially increased by addingpolyanions to preformed DNA/polycation complexes(i.e. recharging). Weconfirmed the same phenomenon for cationic lipids (provisionalapplication Ser. No. 60/150,160).

[0017] In this application we extend this principle into situationswhere DNA-binding polycation and polyanion are covalently linked intoone polymer. Polyanions (polyanion=pA; polycation=pC) of higher chargedensity can displace DNA from its complex with polycation while pAs withlower charge density form triple complexes in which the complexes have anegative surface charge (Y Xu, F C Szoka, Jr., Biochemistry, 35,5616-5623, (1996), V S Trubeiskoy, A Loomis, J E Hagstrom, V G Budker, JA Wolff, Nucleic Acids Res. 27, 3090-3095 (1999)). Similarly, one canexpect formation of DNA/polyampholyte complex in situations where apolyanion block ionically attached to a polyampholyte possesses a chargedensity lower than the charge density of the DNA molecule; A DNAmolecule will be released from a complex with a polyampholyte when apolyanion block has a charge density higher than the DNA molecule (seeFIG. 1). In the latter case, an internal pA-pC salt is formed.

[0018] It has previously been demonstrated that binding ofnegatively-charged serum components can significantly decrease genetransfer efficacy of DNA/polycation (DNA/pC) complexes in vivo (VitielloL, Bockhold K, Joshi P B, Worton P B, Gene Therapy 5, 1306-13 (1998);Ross P C, Hui S W, Gene Therapy 6, 651-659 (1999). We have found thataddition of polyanions to the point of near complex charge reversaldrastically increases the efficacy of gene transfer mediated by DNA/pCcomplex upon i/v administration in mice (Provisional application Ser.No. 60/093,153). This improvement takes place due to protecting effectof pA which is situated as an outside shell on the triple complex andfunctions by inhibiting interactions of the complexes with serumproteins. We believe that gene transfer increase observed withDNA/polyampholyte complexes is based on the same phenomenon. Thepolyanion portion of polyampholyte being displaced from DNA/pCinteraction can form an outside “shell” of negative charge and protectthe complex from inhibiting interactions with serum proteins. The chargedensity of the pA is of primary importance. The higher charge density,the more effective is the protective effect against serum proteins.

[0019] In some cases a polyanionic block may be a natural protein orpeptide used for cell targeting or other function. A polyanionic blockcan provide other functions too: for example, poly(propylacrylic acid)is known for pH-dependent membrane-disruptive function (Murthy N,Robichaud JR, Tirrell D A, Stayton P S, Hoffman A S, Controlled Release(1999) 61:137-43).

[0020] To demonstrate the principle we synthesized two blockpolyampholytes of linear polyethyleneimine (lPEI) with 1)polymethacrylic acid (lPEI-pMAA, high charge density pA) andpolyglutamic acid (lPEI-pGlu, low charge density pA) and preparedcomplexes with plasmid DNA (pCIluc). We show that a covalent complexbetween pC and pA can substantially enhance gene transfer activity whencompared to a non-polyampholyte mixture. We further describe thephenomena in the examples section of this application. In thisspecification, the use of the term polyanion may refer to the anionicportion of the polyampholyte and the term polycation may refer to thecationic portion of the polyampholyte. Abbreviations: Poly-L-Lysine(PLL), succinic anhydride-PLL (SPLL), polymethacrylic acid, pMAA andpolyaspartic acid, pAsp.

[0021] Polymers

[0022] A polymer is a molecule built up by repetitive bonding togetherof smaller units called monomers. In this application the term polymerincludes both oligomers which have two to about 80 monomers and polymershaving more than 80 monomers. The polymer can be linear, branchednetwork, star, comb, or ladder types of polymer. The polymer can be ahomopolymer in which a single monomer is used or can be copolymer inwhich two or more monomers are used. Types of copolymers includealternating, random, block and graft.

[0023] To those skilled in the art of polymerization, there are severalcategories of polymerization processes that can be utilized in thedescribed process. The polymerization can be chain or step. Thisclassification description is more often used that the previousterminology of addition and condensation polymer.

[0024] Step Polymerization:

[0025] In step polymerization, the polymerization occurs in a stepwisefashion. Polymer growth occurs by reaction between monomers, oligomersand polymers. No initiator is needed since there is the same reactionthroughout and there is no termination step so that the end groups arestill reactive. The polymerization rate decreases as the functionalgroups are consumed.

[0026] Typically, step polymerization is done either of two differentways. One way, the monomer has both reactive functional groups (A and B)in the same molecule so that

[0027] A-B yields-(A-B)-

[0028] Or the other approach is to have two difunctional monomers.

[0029] A-A+B-B yields-(A-A-B-B)-

[0030] Generally, these reactions can involve acylation or alkylation.Acylation is defined as the introduction of an acyl group (—COR) onto amolecule. Alkylation is defined as the introduction of an alkyl grouponto a molecule.

[0031] “If functional group A is an amine then B can be (but notrestricted to) an isothiocyanate, isocyanate, acyl azide,N-hydroxysuccinimide, sulfonyl chloride, aldehyde (includingformaldehyde and glutaraldehyde), ketone, epoxide, carbonate,imidoester, carboxylate activated with a carbodiimide, alkylphosphate,arylhalides (difluoro-dinitrobenzene), anhydride, or acid halide,p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester,pentafluorophenyl ester, carbonylimidazole, carbonyl pyridinium, orcarbonyl dimethylaminopyridinium. In other terms when function A is anamine then function B can be acylating or alkylating agent or aminationagent.

[0032] If functional group A is a sulfhydryl then function B can be (butnot restricted to) an iodoacetyl derivative, maleimide, aziridinederivative, acryloyl derivative, fluorobenzene derivatives, or disulfidederivative (such as a pyridyl disulfide or 5 thio-2-nitrobenzoicacid{TNB} derivatives).

[0033] If functional group A is carboxylate then function B can be (butnot restricted to) adiazoacetate or an amine in which a carbodiimide isused. Other additives may be utilized such as carbonyldiimidazole,dimethylamino pyridine (DMAP), N-hydroxysuccinimide or alcohol usingcarbodiimide and DMAP.

[0034] If functional group A is an hydroxyl then function B can be (butnot restricted to) an epoxide, oxirane, or an amine in whichcarbonyldiimidazole or N, N′-disuccinimidyl carbonate, orN-hydroxysuccinimidyl chloroformate or other chloroformates are used. Iffunctional group A is an aldehyde or ketone then function B can be (butnot restricted to) an hydrazine, hydrazide derivative, amine (to form aSchiff Base that may or may not be reduced by reducing agents such asNaCNBH3) or hydroxyl compound to form a ketal or acetal.

[0035] Yet another approach is to have one bifunctional monomer so thatA-A plus another agent yields-(A-A)-. If function A is a sulfhydrylgroup then it can be converted to disulfide bonds by oxidizing agentssuch as iodine (I2) or NaIO4 (sodium periodate), or oxygen (O2).Function A can also be an amine that is converted to a sulfhydryl groupby reaction with 2-Iminothiolate (Traut's reagent) which then undergoesoxidation and disulfide formation. Disulfide derivatives (such as apyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) canalso be used to catalyze disulfide bond formation. Functional group A orB in any of the above examples could also be a photoreactive group suchas aryl azide (including halogenated aryl azide), diazo, benzophenone,alkyne or diazirine derivative.

[0036] Reactions of the amine, hydroxyl, sulfhydryl, carboxylate groupsyield chemical bonds that are described as amide, amidine, disulfide,ethers, esters, enamine, imine, urea, isothiourea, isourea, sulfonamide,carbamate, alkylamine bond (secondaryamine), carbon-nitrogen singlebonds in which the carbon contains a hydroxyl group, thioether, diol,hydrazone, diazo, or sulfone”.

[0037] If functional group A is an aldehyde or ketone then function Bcan be (but not restricted to) an hydrazine, hydrazide derivative, amine(to form a Schiff Base that may or may not be reduced by reducing agentssuch as NaCNBH3) or hydroxyl compound to form a ketal or acetal.

[0038] Yet another approach is to have one difunctional monomer so that

[0039] A-A plus another agent yields-(A-A)-.

[0040] If function A is a sulfhydryl group then it can be converted todisulfide bonds by oxidizing agents such as iodine (I2) or NaIO4 (sodiumperiodate), or oxygen (O2). Function A can also be an amine that isconverted to a sulfhydryl group by reaction with 2-iminothiolate(Traut's reagent) which then undergoes oxidation and disulfideformation. Disulfide derivatives (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid{TNB} derivatives) can also be used tocatalyze disulfide bond formation.

[0041] Functional group A or B in any of the above examples could alsobe a photoreactive group such as aryl azides, halogenated aryl azides,diazo, benzophenones, alkynes or diazirine derivatives.

[0042] Reactions of the amine, hydroxyl sulfhydryl, carboxylate groupsyield chemical bonds that are described as amide, amidine, disulfide,ethers, esters, enamine, urea, isothiourea, isourea, sulfonamnide,carbamate, carbon-nitrogen double bond (imine), alkylamine bond(secondary amine), carbon-nitrogen single bonds in which the carboncontains a hydroxyl group, thio-ether, diol, hydrazone, diazo, orsulfone.

[0043] Chain Polymerization:

[0044] In chain-reaction polymerization growth of the polymer occurs bysuccessive addition of monomer units to limited number of growingchains. The initiation and propagation mechanisms are different andthere is usually a chain-terminating step. The polymerization rateremains constant until the monomer is depleted.

[0045] Monomers containing vinyl, acrylate, methacrylate, acrylamide,methaacrylamide groups can undergo chain reaction which can be radical,anionic, or cationic. Chain polymerization can also be accomplished bycycle or ring opening polymerization. Several different types of freeradical initiatiors could be used that include peroxides, hydroxyperoxides, and azo compounds such as 2,2′-Azobis(-amidinopropane)dihydrochloride (AAP). A compound is a material made up of two or moreelements.

[0046] Types of Monomers:

[0047] A wide variety of monomers can be used in the polymerizationprocesses. These include positive charged organic monomers such asamines, imidine, guanidine, imine, hydroxylamine, hydrozyine,heterocycles (like imidazole, pyridine, morpholine, pyrimidine, orpyrene. The amines could be pH-sensitive in that the pKa of the amine iswithin the physiologic range of 4 to 8. Specific amines includespermine, spermidine, N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD),and 3,3′-Diamino-N,N-dimethyldipropylammonium bromide.

[0048] Monomers can also be hydrophobic, hydrophilic or amphipathic.Amphipathic compounds have both hydrophilic (water-soluble) andhydrophobic (water-insoluble) parts. Hydrophilic groups indicate inqualitative terms that the chemical moiety is water-preferring.Typically, such chemical groups are water soluble, and are hydrogen bonddonors or acceptors with water. Examples of hydrophilic groups includecompounds with the following chemical moieties carbohydrates;polyoxyethylene, peptides, oligonucleotides and groups containingamines, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls.Hydrophobic groups indicate in qualitative terms that the chemicalmoiety is water-avoiding. Typically, such chemical groups are not watersoluble, and tend not to hydrogen bond. Hydrocarbons are hydrophobicgroups. Monomers can also be intercalating agents such as acridine,thiazole organge, or ethidium bromide.

[0049] Other Components of the Monomers and Polymers:

[0050] The polymers have other groups that increase their utility. Thesegroups can be incorporated into monomers prior to polymer formation orattached to the polymer after its formation. These groups include:Targeting Groups-such groups are used for targeting the polymer-nucleicacid complexes to specific cells or tissues. Examples of such targetingagents include agents that target to the asialoglycoprotein receptor byusing asiologlycoproteins or galactose residues. Other proteins such asinsulin, EGF, or transferrin can be used for targeting. Protein refersto a molecule made up of 2 or more amino acid residues connected one toanother as in a polypeptide. The amino acids may be naturally occurringor synthetic. Peptides that include the RGD sequence can be used totarget many cells. Peptide refers to a linear series of amino acidresidues connected to one another by peptide bonds between thealpha-amino group and carboxyl group of contiguous amino acid residues.Chemical groups that react with sulfhydryl or disulfide groups on cellscan also be used to target many types of cells. Folate and othervitamins can also be used for targeting. Other targeting groups includemolecules that interact with membranes such as fatty acids, cholesterol,dansyl compounds, and amphotericin derivatives.

[0051] After interaction of the supramolecular complexes with the cell,other targeting groups can be used to increase the delivery of the drugor nucleic acid to certain parts of the cell. For example, agents can beused to disrupt endosomes and a nuclear localizing signal (NLS) can beused to target the nucleus.

[0052] A variety of ligands have been used to target drugs and genes tocells and to specific cellular receptors. The ligand may seek a targetwithin the cell membrane, on the cell membrane or near a cell. Bindingof ligands to receptors typically initiates endocytosis. Ligands couldalso be used for DNA delivery that bind to receptors that are notendocytosed. For example peptides containing RGD peptide sequence thatbind integrin receptor could be used. In addition viral proteins couldbe used to bind the complex to cells. Lipids and steroids could be usedto directly insert a complex into cellular membranes.

[0053] The polymers can also contain cleavable groups within themselves.When attached to the targeting group, cleavage leads to reduceinteraction between the complex and the receptor for the targetinggroup. Cleavable groups include but are not restricted to disulfidebonds, diols, diazo bonds, ester bonds, sulfone bonds, acetals, ketals,enol ethers, enol esters, enamines and imines.

[0054] Reporter or marker molecules are compounds that can be easilydetected Typically they are fluorescent compounds such as fluorescein,rhodamine, texas red, CY-5, CY-3 or dansyl compounds. They can bemolecules that can be detected by UV or visible spectroscopy or byantibody interactions or by electron spin resonance. Biotin is anotherreporter molecule that can be detected by labeled avidin. Biotin couldalso be used to attach targeting groups.

[0055] A polycation is a polymer containing a net positive charge, forexample poly-L-lysine hydrobromide. The polycation can contain monomerunits that are charge positive, charge neutral, or charge negative,however, the net charge of the polymer must be positive. A polycationalso can mean a non-polymeric molecule that contains two or morepositive charges. A polyanion is a polymer containing a net negativecharge, for example polyglutamic acid. The polyanion can contain monomerunits that are charge negative, charge neutral, or charge positive,however, the net charge on the polymer must be negative. A polyanion canalso mean a non-polymeric molecule that contains two or more negativecharges. The term polyion includes polycation, polyanion, zwitterionicpolymers, and neutral polymers. The term zwitterionic refers to theproduct (salt) of the reaction between an acidic group and a basic groupthat are part of the same molecule. Salts are ionic compounds thatdissociate into cations and anions when dissolved in solution. Saltsincrease the ionic strength of a solution, and consequently decreaseinteractions between nucleic acids with other cations. A charged polymeris a polymer that contains residues, monomers, groups, or parts with apositive or negative charge and whose net charge can be neutral,positive, or negative.

[0056] Signals

[0057] In a preferred embodiment, a chemical reaction can be used toattach a signal to a nucleic acid complex. The signal is defined in thisspecification as a molecule that modifies the nucleic acid complex andcan direct it to a cell location (such as tissue cells) or location in acell (such as the nucleus) either in culture or in a whole organism. Bymodifying the cellular or tissue location of the foreign gene, theexpression of the foreign gene can be enhanced.

[0058] The signal can be a protein, peptide, lipid, steroid, sugar,carbohydrate, nucleic acid or synthetic compound The signals enhancecellular binding to receptors, cytoplasmic transport to the nucleus andnuclear entry or release from endosomes or other intracellular vesicles.

[0059] Nuclear localizing signals enhance the targeting of the gene intoproximity of the nucleus and/or its entry into the nucleus. Such nucleartransport signals can be a protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear localizing signalsinteract with a variety of nuclear transport factors such as the NLSreceptor (karyopherin alpha) which then interacts with karyopherin beta.The nuclear transport proteins themselves could also function as NLS'ssince they are targeted to the nuclear pore and nucleus.

[0060] Signals that enhance release from intracellular compartments(releasing signals) can cause DNA release from intracellularcompartments such as endosomes (early and late), lysosomes, phagosomes,vesicle, endoplasmic reticulum, Golgi apparatus, trans Golgi network(TGN), and sarcoplasmic reticulum. Release includes movement out of anintracellular compartment into cytoplasm or into an organelle such asthe nucleus. Releasing signals include chemicals such as chloroquine,bafilomycin or Brefeldin A1 and the ER-retaining signal (KDEL sequence),viral components such as influenza virus hemagglutinin subunit HA-2peptides and other types of amphipathic peptides.

[0061] Cellular receptor signals are any signal that enhances theassociation of the gene or particle with a cell. This can beaccomplished by either increasing the binding of the gene to the cellsurface and/or its association with an intracellular compartment, forexample: ligands that enhance endocytosis by enhancing binding the cellsurface. This includes agents that target to the asialoglycoproteinreceptor by using asiologlycoproteins or galactose residues. Otherproteins such as insulin, EGF, or transferrin can be used for targeting.Peptides that include the RGD sequence can be used to target many cells.Chemical groups that react with sulfhydryl or disulfide groups on cellscan also be used to target many types of cells. Folate and othervitamins can also be used for targeting. Other targeting groups includemolecules that interact with membranes such as lipids fatty acids,cholesterol, dansyl compounds, and amphotericin derivatives. In additionviral proteins could be used to bind cells.

[0062] Other Definitions:

[0063] Extravascular means outside of a vessel such as a blood vessel.Extravascular space means an area outside of a vessel. Space may containbiological matter such as cells and does not imply empty space.

[0064] Extravasation means the escape of material such as compounds andcomplexes from the vessel into which it is introduced into theparenchymal tissue or body cavity.

[0065] The process of delivering a polynucleotide to a cell has beencommonly termed “transfection” or the process of “transfecting” and alsoit has been termed “transformation”. The polynucleotide could be used toproduce a change in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic and researchpurposes is commonly called “gene therapy”. The polynucleotides orgenetic material being delivered are generally mixed with transfectionreagents prior to delivery.

[0066] The polyampholyte complex is a complex having the potential toreact with biological components. More particularly, polyampholytecomplexes utilized in this specification are designed to change thenatural processes associated with a living cell. For purposes of thisspecification, a cellular natural process is a process that isassociated with a cell before delivery of a polyamtpholyte complex. Inthis specification, the cellular production of, or inhibition of amaterial, such as a protein, caused by a human assisting a molecule toan in vivo cell is an example of a delivered biologically activecompound. Pharmaceuticals, proteins, peptides, polypeptides, hormones,cytokines, antigens, viruses, oligonucleotides, and nucleic acids areexamples that can be components of polyampholyte complexes.

[0067] The term “nucleic acid” is a term of art that refers to a polymercontaining at least two nucleotides. “Nucleotides” contain a sugardeoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and syntheticderivatives of purines and pyrimidines, or natural analogs. Nucleotidesare the monomeric units of nucleic acid polymers. A “polynucleotide” isdistinguished here from an “oligonucleotide” by containing more than 80monomeric units; oligonucleotides contain from 2 to 80 nucleotides. Theterm nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). DNA may be in the form of antisense, plasmid DNA, parts of aplasmid DNA, vectors (Pl, PAC, BAC, YAC, artificial chromosomes),expression cassettes, chimeric sequences, chromosomal DNA, orderivatives of these groups. RNA may be in the form of oligonucleotideRNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomalRNA), mRNA (messenger RNA), anti-sense RNA, ribozymes, chimericsequences, or derivatives of these groups. “Anti-sense” is apolynucleotide that interferes with the function of DNA and/or RNA. Thismay result in suppression of expression. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones, nucleotides, or bases. These include PNAs (peptide nucleicacids), phosphothionates, and other variants of the phosphate backboneof native nucleic acids. In addition, DNA and RNA may be single, double,triple, or quadruple stranded. “Expression cassette” refers to a naturalor recombinantly produced polynucleotide molecule which is capable ofexpressing protein(s). A DNA expression cassette typically includes apromoter (allowing transcription initiation), and a sequence encodingone or more proteins. Optionally, the expression cassette may includetranscriptional enhancers, non-coding sequences, splicing signals,transcription termination signals, and polyadenylation signals. An RNAexpression cassette typically includes a translation initiation codon(allowing translation initiation), and a sequence encoding one or moreproteins. Optionaly, the expression cassette may include translationtermination signals, a polyadenosine sequence, internal ribosome entrysites (IRES), and non-coding sequences.

[0068] The term “naked polynucleotides” indicates that thepolynucleotides are not associated with a transfection reagent or otherdelivery vehicle that is required for the polynucleotide to be deliveredto the cardiac muscle cell. A transfection reagent is a compound orcompounds used in the prior art that bind(s) to or complex(es) witholigonucleotides and polynucleotides, and mediates their entry intocells. The transfection reagent also mediates the binding andinternalization of oligonucleotides and polynucleotides into cells.Examples of transfection reagents include cationic liposomes and lipids,polyamines, calcium phosphate precipitates, histone proteins,polyethylenimine, and polylysine complexes. It has been shown thatcationic proteins like histones and protamines, or synthetic polymerslike polylysine, polyarginine, polyornithine, DEAE dextran, polybrene,and polyethylenimine may be effective intracellular delivery agents,while small polycations like spermine may be ineffective. Typically, thetransfection reagent has a net positive charge that binds to theoligonucleotide's or polynucleotide's negative charge. The transfectionreagent mediates binding of oligonucleotides and polynucleotides tocells via its positive charge (that binds to the cell membrane'snegative charge) or via ligands that bind to receptors in the cell. Forexample, cationic liposomes or polylysine complexes have net positivecharges that enable them to bind to DNA or RNA. Polyethylenimine, whichfacilitates gene expression without additional treatments, probablydisrupts endosomal function itself.

[0069] Other vehicles are also used, in the prior art, to transfer genesinto cells. These include complexing the polynucleotides on particlesthat are then accelerated into the cell. This is termed “biolistic” or“gun” techniques. Other methods include “electroporation,” in which adevice is used to give an electric charge to cells. The charge increasesthe permeability of the cell.

[0070] Charge density is the term used to describe the electrical chargeper unit area, for example, on a polymer.

[0071] Ionic (electrostatic) interactions are the non-covalentassociation of two or more substances due to attractive forces betweenpositive and negative charges, or partial positive and partial negativecharges.

[0072] Condensed Nucleic Acids:

[0073] Condensing a polymer means decreasing the volume that the polymeroccupies. An example of condensing nucleic acid is the condensation ofDNA that occurs in cells. The DNA from a human cell is approximately onemeter in length but is condensed to fit in a cell nucleus that has adiameter of approximately 10 microns. The cells condense (or compacts)DNA by a series of packaging mechanisms involving the histones and otherchromosomal proteins to form nucleosomes and chromatin. The DNA withinthese structures is rendered partially resistant to nuclease DNase)action. The process of condensing polymers can be used for deliveringthem into cells of an organism.

[0074] A delivered polymer can stay within the cytoplasm or nucleusapart from the endogenous genetic material. Alternatively, the polymercould recombine (become a part of) the endogenous genetic material. Forexample, DNA can insert into chromosomal DNA by either homologous ornon-homologous recombination.

[0075] Condensed nucleic acids may be delivered intravasculary,intrarterially, intravenously, orally, intraduodenaly, via the jejunum(or ileum or colon), rectally, transdermally, subcutaneously,intramuscularly, intraperitoneally, intraparenterally, via directinjections into tissues such as the liver, lung, heart, muscle, spleen,pancreas, brain (including intraventricular), spinal cord, ganglion,lymph nodes, lymphatic system, adipose tissues, thyroid tissue, adrenalglands, kidneys, prostate, blood cells, bone marrow cells, cancer cells,tumors, eye retina, via the bile duct, or via mucosal membranes such asin the mouth, nose, throat, vagina or rectum or into ducts of thesalivary or other exocrine glands. “Delivered” means that thepolynucleotide becomes associated with the cell. The polynucleotide canbe on the membrane of the cell or inside the cytoplasm, nucleus, orother organelle of the cell.

[0076] An intravascular route of administration enables a polymer orpolynucleotide to be delivered to cells more evenly distributed and moreefficiently expressed than direct injections. Intravascular herein meanswithin a tubular structure called a vessel that is connected to a tissueor organ within the body. Within the cavity of the tubular structure, abodily fluid flows to or from the body part. Examples of bodily fluidinclude blood, lymphatic fluid, or bile. Examples of vessels includearteries, arterioles, capillaries, venules, sinusoids, veins,lymphatics, and bile ducts. The intravascular route includes deliverythrough the blood vessels such as an artery or a vein.

[0077] An administration route involving the mucosal membranes is meantto include nasal, bronchial, inhalation into the lungs, or via the eyes.

[0078] Recharging Condensed Nucleic Acids

[0079] Polyions for gene therapy and gene therapy research can involveanionic systems as well as charge neutral or charge-positive systems.The ionic polymer can be utilized in “recharging” (another layer havinga different charge) the condensed polynucleotide complex. The resultingrecharged complex can be formed with an appropriate amount of chargesuch that the resulting complex has a net negative, positive or neutralcharge. The interaction between the polycation and the polyanion can beionic, can involve the ionic interaction of the two polymer layers withshared cations, or can be crosslinked between cationic and anionic siteswith a crosslinking system (including cleavable crosslinking systems,such as those containing disulfide bonds). The interaction between thecharges located on the two polymer layers can be influenced with the useof added ions to the system. With the appropriate choice of ion, thelayers can be made to disassociate from one another as the ion diffusesfrom the complex into the cell in which the concentration of the ion islow (use of an ion gradient).

[0080] Electrostatic complexes between water-soluble polyelectrolyteshave been studied widely in recenty ears. Complexes containing DNA as apolyanionic constituent only recently came to the attention because oftheir potential use in gene therapy applications such as non-viral genetransfer preparations (polyplexes) for particle delivery to a cell.Strong polyelectrolytes, polyanion/polycation complexes, are usuallyformed at a 1:1 charge stoichiometrically. A charge ratio 1:1 complexbetween DNA and Poly-L-Lysine (PLL) also has been demonstrated in theprior art.

[0081] Polyanions effectively enhance the gene delivery/gene expressioncapabilities of all major classes of polycation gene delivery reagents.In that regard, we disclose the formation of negatively charged tertiarycomplexes containing nucleic acid, PLL, and succinic anhydride-PLL(SPLL) complexes. SPLL is added to a cationic nucleic acid/PLL complexin solution. Nucleic acid at the core of such complexes remainscondensed, in the form of particles˜50 nm in diameter. DNA and PLL bindsSPLL in 1:1:1 complex with SPLL providing a net negative charge to theentire complex. Such small negatively charged particles are useful fornon-viral gene transfer applications.

[0082] One of the advantages that flow from recharging DNA particles isreducing their non-specific interactions with cells and serum proteins((Wolfert et al. Hum. Gene Therapy 7:2123-2133 (1996); Dash et al., GeneTherapy 6:643-650 (1999); Plank et al., Hum. Gene Ther. 7:1437-1446(1996); Ogris et al., Gene Therapy 6:595-605 (1999); Schacht et al.Brit. Patent Application 9623051.1 (1996)).

[0083] A wide a variety of polyanions can be used to recharge theDNA/polycation particles. They include (but not restricted to): Anywater-soluble polyanion can be used for recharging purposes includingsuccinylated PLL, succinylated PEI (branched), polyglutamic acid,polyaspartic acid, polyacrylic acid, polymethacrylic acid,polyethylacrylic acid, polypropylacrylic acid, polybutylacrylic acid,polymaleic acid, dextran sulfate, heparin, hyaluronic acid,polysulfates, polysulfonates, polyvinyl phosphoric acid, polyvinylphosphonic acid, copolymers of polymaleic acid, polyhydroxybutyric acid,acidic polycarbohydrates, DNA, RNA, negatively charged proteins,pegylated derivatives of above polyanions, pegylated derivativescarrying specific ligands, block and graft copolymers of polyanions andany hydrophilic polymers (PEG, poly(vinylpyrrolidone), poly(acrylamide),etc).

[0084] DNA condensation assays based on the effect ofconcentration-dependent self-quenching of covalently-bound fluorophoresupon DNA collapse indicated essentially the same phenomenon described inthe prior art. Polyanions with high charge density (polymethacrylicacid, pMAA and polyaspartic acid, pAsp) were able to decondense DNAprior to those that complexed with PLL while polyanions with lowercharge density (polyglutamic acid, pGlu, SPLL) failed to decondense DNA.Together with z-potential measurements, these data represent support forthe presence of negatively charged condensed DNA particles. Theseparticles are approximately 50 nm in diameter in low salt buffer asmeasured by atomic force microscopy which revealed particles of spheroidmorphology. This places them very close in size to binary DNA/PLLparticles. Particles prepared using various pC/pA polyampholytes can beused to form similar condensed DNA particles.

[0085] In another preferred embodiment, the polyanion can be covalentlyattached to the polycation using a variety of chemical reactions withoutthe use of crosslinker. The polyanion can contain reactive groups thatcovalently attach to groups on the polycation. This results in theformation of a polyampholyte The types of reactions are similar to thosediscussed above in the section on step polymerization.

[0086] In another preferred embodiment the attachment of the rechargedcomplex can be enhanced by using chelators and crown ethers, preferablypolymeric.

[0087] In one preferred embodiment the DNA/polycation complexes areinitially formed by adding only a small excess of polycation to nucleicacid (in charge ratio which is defined as ratio of polycation totalcharge to polyanion total charge at given pH). The charge ratio ofpolycation to nucleic acid charge could be less than 2, less than 1.7,less than 1.5 or even less than 1.3. This would be preferably done inlow ionic strength solution so as to avoid the complexes fromflocculation. Low ionic strength solution means solution with totalmonovalent salt concentration less than 50 mM. Then the polyanion isadded to the mixture and only a small amount of “blank” particles areformed. “Blank” particles are particles that contain only polycation andpolyanion and no nucleic acid.

[0088] In another preferred embodiment, the polycation is added to thenucleic acid in charge excess but the excess polycation that is not incomplex with the nuclei acid is removed by purificaton. Purificationmeans removing of charged polymer using centrifugation, dialysis,chromatography, electrophoresis, precipitation, extraction.

[0089] Yet in another preferred embodiment a ultracentrifugationprocedure (termed “centrifugation step”) is used to reduce the amount ofexcess polycation, polyanion, or “blank” particles. The method is basedon the phenomenon that only dense DNA-containing particles can becentrifuged through 10% sucrose solution at 25,000 g. Aftercentrifugation purified complex is at the bottom of the tube whileexcess of polyanion and “blank” particles stay on top. In modificationof this experiment 40% solution of metrizamide can be used as a cushionto collect purified DNA/polycation/polyanion complex on the boundary foreasy retrieval.

[0090] The attachment of the polyanion to the DNA/polycation complexenhance stability but can also enable a ligand or signal to be attachedto the DNA particle. This is accomplished by attaching the ligand orsignal to the polyanion which in turn is attached to the DNA particle. Adialysis step or centifugation step can be used to reduce the amount offree polyanion containing a ligand or signal that is in solution and notcomplexed with the DNA particle. One approach is to replace the free,uncomplexed polyanion containing a ligand or signal with free polyanionthat does not contain a ligand or signal.

[0091] Yet in another preferred embodiment a polyanion used for chargereversal is modified with neutral hydrophilic polymer for stericstabilization of the whole complex. The complex formation of DNA withpegylated polycations results in substantial stabilization of thecomplexes towards salt- and serum-induced flocculation (Wolfert et al.Hum. Gene Therapy 7:2123-2133 (1996), Ogris et al., Gene Therapy6:595-605 (1999). We have demonstrated that modification of polyanion intriple complex also significantly enhances salt and serum stability.

[0092] In another preferred embodiment a polyanion used for chargereversal is cleavable. One can imagine two ways to design a cleavablepolyion: 1. A polyion cleavable in backbone, 2. A polyion cleavable inside chain. First scenario would comprise monomers linked by labilebonds such as disulfide, diols, diazo, ester, sulfone, acetal, ketal,enol ether, enol ester, imine and enamine bonds. Second scenario wouldinvolve reactive groups (i.e. electrophiles and nucleophiles) in closeproximity so that reaction between them is rapid. Examples includehaving corboxylic acid derivatives (acids, esters and amides) andalcohols, thiols, carboxylic acids or amines in the same moleculereacting together to make esters, thiol esters, anhydrides or amides. Inone specific preferred embodiment the polyion contains an ester acidsuch as citraconnic acid, or dimethylmaleyl acid that is connected to acarboxylic, alcohol, or amine group on the polyion.

[0093] Cleavable means that a chemical bond between atoms is broken.Labile also means that a chemical bond between atoms is breakable.Crosslinking refers to the chemical attachment of two or more moleculeswith a bifunctional reagent A bifunctional reagent is a molecule withtwo reactive ends. The reactive ends can be identical as in ahomobifunctional molecule, or different as in a heterobifunctionalmolecule.

EXAMPLES Example 1

[0094] Synthesis of lPEI-pMAA and lPEI-pGlu Complexes.

[0095] The following polyions were used for the reaction: lPEI (Mw=25kDa, Polysciences), pMAA (Mw=9.5 kDa, Aldrich), pGlu (Mw=49 kDa, Sigma).For analytical purposes pAs covalently labeled withrhodamine-ethylenediamine (Molecular Probes) were used for thesereactions (degree of carboxy group modification <2%). Absorbance of thepAs was used to trace pAs and conjugates during size exclusionchromatography. PMAA (or pGlu, 1 mg in 100 μL water) was activated inthe presence of water-soluble carbidiimide (EDC, 100 μg) andN-hydroxysulfosuccinimide (100 μg) for 10 min at pH 5.5. Activated pMAAwas added to the solution of lPEI (2 mg in 200 μL of 25 mM HEPES, pH8.0) and incubated for 1 hr at room temperature.

Example 2

[0096] Separation of lPEI-pMAA and lPEI-pGlu Reaction Mixtures usingSepharose 4B-CL Column in 1.5 M NaCl.

[0097] After the reaction completion equal volume of 3 M NaCl solutionwas added to the part of the reaction mixture. This part (0.5 ml) waspasses through the Sepharose 4B-CL column (1×25 cm) equlibrated in 1.5 MNaCl. Volume of the fractions collected was 1 ml. Rhodamine fluorescencewas measured in each fraction. Linear PEI was measured usingfluorescamine reaction. The amount of polyampholyte in the lPEI-pGlureaction mixture is about 50% (see FIG. 2).

Example 3

[0098] HUH7 Mouse Liver Cell Transfection using DNA/lPEI-pAPolyampholyte Mixtures.

[0099] Part of the polyampholyte reaction mixtures lPEI-pMAA andlPEI-pGlu were used to transfect HUH7 cells in culture. Non-covalentmixtures of lPEI with pMAA and pGlu mixed in the same ratios as forconjugates were used as the controls. Luciferase-encoded plasmid pCIluc(2 μg per 35 mm well) was used for transfection in OPTIMEM (cell medium)and OPTIMEM supplemented with 10% bovine serum. Amount of polyampholyteadded is indicated on the basis of lPEI content Results of thisexperiment are shown on FIGS. 3 and 4. There is a strong enhancement oftransfection for polyampholytes in case of weaker pA conjugate(lPEI-pGlu, FIG. 3.) and almost no difference in transfection abilitiesof conjugates and mixtures for stronger pA (lPEI-pMAA, FIG. 4) inaccordance to FIG. 1 scheme.

Example 4

[0100] Optimized Synthesis of lPEI-pGlu Polyampholyte

[0101] Rhodamine-labeled polyglutamic acid (pGlu, 150 uL, 20 mg/ml,titrated to pH 5.0) was activated with water-soluble[3′-(dimethylaminopropyl)-3-ethyl]carbodiimide (EDC, 15 ul, 100 mg/ml inDMSO) and sulfosuccinimide (SNHS, 15 um, 100 mg/ml in water) for 10 min.Then linear PEI (lPEI, 150 ul, 20 mg/ml) was added to the mixture, pHwas adjusted to 8.0 and the mixture was allowed to stand for 2 hrs atroom temperature. After this the mixture was passed through Sepharose4B-CL column (1×20 cm) equilibrated with 1.5 M NaCl solution (FIG. 5 ).Rhodamine fluorescence was measured in each fraction. Fractions 10-14were pooled, dialysed against water and freeze-dried to yield purifiedpolyampholyte.

Example 5

[0102] HUH7 Mouse Liver Cell Transfection using DNA/lPEI-pAPolyampholyte Mixtures in the Presence of 100% Serum.

[0103] The luciferase encoding plasmid pCILuc was used for in vitro andin vivo gene transfer experiments. The DNA/polymer complexes were formedin 5 mM HEPES, 50 mM NaCl, 0.29 M glucose, pH 7.5 at DNA concentrationof 50 ug/ml. HUH7 mouse liver cells were subconfluently seeded in12-well plates. The complexes (1 ug of DNA) were added directly to 1 mlof 100% bovine serum into each well and incubated with cells for 4 hrs.After this step the cells were washed with OPTI-MEM media, supplementedwith fresh media and maintained for additional 48 hrs. After this periodof time the cells were harvested, lysed and processed for luciferaseexpression measurements. Non-covalent mixture of lPEI and pGlu as wellas lPEI alone were used a controls in this experiment (FIG. 6). As onecan see, the covalent conjugate of lPEI and pGlu gave significantlyhigher gene transfer activity in high range of polymer/DNA ratios ascompared to controls.

Example 6

[0104] Synthesis of Branched PEI (brPEI)-pGlu and brPEI-pAspPolyampholytes

[0105] Polyglutamic acid (pGlu, 2.28 mg in 172 ul of water, pH 5.5) orpolyaspartic acid (pAsp, 2 mg in 172 ul of water) were activated in thepresence of 100 ug of EDC and SNHS each for 10 min at room temperature.BrPEI (4 mg) and 2.5 M Na Cl (0.5 ml) solutions were added to theactivated polyanion. The reaction mixture was allowed to incubate for 5hrs at room temperature. Resulting brPEI-based polyampholytes weredialyzed against water and freeze-dried.

Example 7

[0106] In vivo Gene Transfer Activity of DNA/Polyampholyte ComplexesPrepared from Branched PEI.

[0107] BrPEI-pGlu and brPEI-pAsp polyampholytes were mixed with DNA atdifferent w/w ratios in 5 mM HEPES, 0.29 M glucose, pH 7.5 at the DNAconcentration of 0.2 mg/ml. The complexes (0.25 ml per animal) wereintravenously injected into mouse tail vein (2 animals per group). Theanimals were sacrificed 24 hrs after injection and the lungs wereprocessed for luciferase activity. The results of in vivo gene transferare presented in Table 1: DNA/brPEI- DNA/brPEI- Ratio (w/w) DNA/brPEIpAsp pGlu 1:1 600, non-toxic 88,000, non- 34,000, non- toxic toxic 1:2All died 600,000, one 3,900,000, died non-toxic 1:3 n/a n/a 4,800,000,one died

[0108] Table 1.

[0109] Luciferase activity (RLU) in lungs after intravenousadministration of DNA/brPEI-based polyampholytes in mice. Each animalreceived 50 ug of DNA in 0.25 ml of isotonic glucose solution. Therewere 2 animals per group. Survival of all animals in the group marked asnon-toxic.

[0110] As one can see, complexing DNA with brPEI-based polyampholytesresults in effective preparations for DNA delivery to parenchymal cells.BrPEI alone is ineffective at low weight ratios and toxic at higherratios. Covalent conjugation of polyanions results in significantincrease in gene transfer efficacy in lungs accompanying with reductionof toxicity.

[0111] The foregoing is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will readily occur to those skilled in the art it is not desiredto limit the invention to the exact construction and operation shown anddescribed. Therefore, all suitable modifications and equivalents fallwithin the scope of the invention.

We claim:
 1. A process for enhancing delivery of a polyion to a cel,comprising: a) forming a complex of polyampholyte and polyion; and, b)delivering the complex into a cell.
 2. The process of claim 1 whereinthe polyampholyte comprises a polycation selected from group consistingof PLL and PEI.
 3. The process of claim 1 wherein the polyampholytecomprises a polyanion.
 4. The process of claim 3 wherein the polyanioncomprises a molecule selected from the group consisting of succinylatedPLL, succinylated PEI, polyglutamic acid, polyaspartic acid, polyacrylicacid, polymethacrylic acid, dextran sulfate, heparin, hyaluronic acid,DNA, RNA, and negatively charged proteins.
 5. The process of claim 3wherein the polyanion comprises a molecule selected from the groupconsisting of pegylated derivatives, pegylated derivatives carryingspecific ligands, block copolymers, graft copolymers and hydrophilicpolymers.
 6. The process of claim 1 wherein the polyampholyte isdelivered to a cell in vivo.
 7. A complex for delivering a polyion to acell, comprising: a) a polyion; and, b) a polyampholyte wherein thepolyion and the polyampholyte are bound in complex.
 8. The complex ofclaim 7 wherein the polyampholyte comprises a polycation.
 9. The complexof claim 8 wherein the polycation is selected from group consisting ofPLL, PEI histones or cationic lipids.
 10. The complex of claim 7 whereinthe polyampholyte comprises a polyanion.
 11. The complex of claim 10wherein the polyanion comprises a molecule selected from the groupconsisting of succinylated PLL, succinylated PEI, polyglutamic acid,polyaspartic acid, polyacrylic acid, polymethacrylic acid, dextransulfate, heparin, hyaluronic acid, DNA, RNA, and negatively chargedproteins.
 12. The complex of claim 11 wherein the polyanion comprises amolecule selected from the group consisting of pegylated derivatives,pegylated derivatives carrying specific ligands, block copolymers, graftcopolymers and hydrophilic polymers.
 13. A process for extravasation ofa complex, comprising: a) forming a complex of polyampholyte andpolyion; and, b) inserting the complex into a vessel; c) delivering thecomplex to an extravascular space.
 14. The complex of claim 13 whereinthe polyampholyte comprises a polycation which is selected from groupconsisting of PLL PEI, histones or cationic lipids.
 15. The complex ofclaim 13 wherein the polyampholyte comprises a polyanion selected fromthe group consisting of succinylated PLL, succinylated PEI, polyglutamicacid, polyaspartic acid, polyacrylic acid, polymethacrylic acid, dextransulfate, heparin, hyaluronic acid, DNA, RNA, and negatively chargedproteins.
 16. The complex of claim 15 wherein the negatively chargedpolyion comprises a molecule selected from the group consisting ofpegylated derivatives, pegylated derivatives carrying specific ligands,block copolymers, graft copolymers and hydrophilic polymers.
 17. Theprocess of claim 13 wherein the complex is delivered to an extravascularcell.
 18. The process of claim 13 wherein the polyion consists of anucleic acid.
 19. The process of claim 18 wherein the nucleic acid isexpressed.